Active and secreted IgA-coated bacterial fractions from the human gut reveal an under-represented

Transcription

1 Supplemental information Title. Active and secreted IgA-coated bacterial fractions from the human gut reveal an under-represented microbiota core. Authors. Giuseppe D'Auria, Francesc Peris-Bondia, Mária Džunková, Alex Mira, Maria Carmen Collado, Amparo Latorre and Andrés Moya

2 Table 1: Analysis of variance using distance matrix (Adonis). The F-test is based on sequential sums of squares from permutations of the raw data, and not permutations of residuals. Analysis was carried out using environmental metadata grouping data by fractions (FS, Act and IgA) and samples (V1, V2, V3, V4, V5 and V6). df Sums of squares Mean square F statistic R2 Pr(>F) FRACTION ** SAMPLE ** Residuals Total

3 Table 2: Multiplex identifiers to tag each PCR reaction. Same MIDs have been used in different pyrosequencing runs. MID Sequence SAMPLE MID1 TCGCTTCCTATC V1.Iga, V1.Act, V1.Fs MID2 TCTAATGCGCTC V2.IgA, V2.Act, V2.FS MID3 TCGCTGCTTGTC V3.IgA, V3.Act, V3.FS MID4 TCTACGAACCTC V4.IgA, V4.Act, V4.FS MID5 TCGGATAACGTC V5.IgA, V5.Act, V5.FS MID6 TCTAGCGGAATC V6.IgA, V6.Act, V6.FS

4 Figure 1: All/unidentified genus distribution. Bar charts show the distributions of all and unidentified genera (as sum) among all samples and fractions. Blue gradients describe all genus frequencies within phyla. Red gradients show the proportion of unidentified genera. Data are represented in logarithmic scale. Groups of bar charts describe distributions among phyla (X axis). Each group describes distributions among fractions FS, Act and IgA respectively (see legend on top). Only major phyla are represented.

5 Figure 2: Bar charts describe the distribution of reads at genus level for main phyla. For each phylum every chart refers to a sample (headers). Bars represent frequency values expressed in percentage. Act and IgA values have been corrected taking into account the representativeness of the fraction with respect to the total number of cells (see Materials and Methods section). Y axis data are reported in percentage and on a logarithmic scale in order to highlight under-represented bacteria.

6 Phylum Class Order Family Genus Firmicutes Act FS IgA V6 V5 V4 Frequency (Log scale) V3 V2 V1 Firmicutes Bacilli Bacillales Bacillaceae 1 Bacillus Firmicutes Bacilli Bacillales Bacillaceae 1 Geobacillus Firmicutes Bacilli Bacillales Bacillales_Incertae Sedis XI Gemella Firmicutes Bacilli Bacillales Planococcaceae Planococcus Firmicutes Bacilli Bacillales Staphylococcaceae Salinicoccus Firmicutes Bacilli Bacillales Staphylococcaceae Staphylococcus Firmicutes Bacilli Bacillales Staphylococcaceae ud Staphylococcaceae Firmicutes Bacilli Bacillales ud Bacillales ud Bacillales Firmicutes Bacilli Lactobacillales Aerococcaceae Aerococcus Firmicutes Bacilli Lactobacillales Carnobacteriaceae Granulicatella Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus Firmicutes Bacilli Lactobacillales Enterococcaceae ud Enterococcaceae Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus Firmicutes Bacilli Lactobacillales Lactobacillaceae ud Lactobacillaceae Firmicutes Bacilli Lactobacillales Leuconostocaceae Leuconostoc Firmicutes Bacilli Lactobacillales Leuconostocaceae Weissella Firmicutes Bacilli Lactobacillales Streptococcaceae Lactococcus Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus Firmicutes Bacilli Lactobacillales Streptococcaceae ud Streptococcaceae Firmicutes Bacilli Lactobacillales ud Lactobacillales ud Lactobacillales Firmicutes Bacilli ud Bacilli ud Bacilli ud Bacilli Firmicutes Clostridia Clostridiales Clostridiaceae 1 Anaerobacter Firmicutes Clostridia Clostridiales Clostridiaceae 1 Clostridium sensu stricto Firmicutes Clostridia Clostridiales Clostridiaceae 1 ud Clostridiaceae 1 Firmicutes Clostridia Clostridiales Clostridiales_Incertae Sedis XIII Anaerovorax Firmicutes Clostridia Clostridiales Clostridiales_Incertae Sedis XIII Mogibacterium Firmicutes Clostridia Clostridiales Clostridiales_Incertae Sedis XIII ud Clostridiales_Incertae Sedis XIII Firmicutes Clostridia Clostridiales Lachnospiraceae Anaerostipes Firmicutes Clostridia Clostridiales Lachnospiraceae Blautia Firmicutes Clostridia Clostridiales Lachnospiraceae Butyrivibrio Firmicutes Clostridia Clostridiales Lachnospiraceae Clostridium XlVa Firmicutes Clostridia Clostridiales Lachnospiraceae Clostridium XlVb Firmicutes Clostridia Clostridiales Lachnospiraceae Coprococcus Firmicutes Clostridia Clostridiales Lachnospiraceae Dorea Firmicutes Clostridia Clostridiales Lachnospiraceae Hespellia Firmicutes Clostridia Clostridiales Lachnospiraceae Howardella Firmicutes Clostridia Clostridiales Lachnospiraceae Lachnospiracea_incertae_sedis Firmicutes Clostridia Clostridiales Lachnospiraceae Lactonifactor Firmicutes Clostridia Clostridiales Lachnospiraceae Oribacterium Firmicutes Clostridia Clostridiales Lachnospiraceae Pseudobutyrivibrio Firmicutes Clostridia Clostridiales Lachnospiraceae Roseburia Firmicutes Clostridia Clostridiales Lachnospiraceae Syntrophococcus Firmicutes Clostridia Clostridiales Lachnospiraceae ud Lachnospiraceae Firmicutes Clostridia Clostridiales Peptostreptococcaceae Clostridium XI Firmicutes Clostridia Clostridiales Peptostreptococcaceae ud Peptostreptococcaceae Firmicutes Clostridia Clostridiales Ruminococcaceae Anaerotruncus Firmicutes Clostridia Clostridiales Ruminococcaceae Butyricicoccus Firmicutes Clostridia Clostridiales Ruminococcaceae Clostridium IV Firmicutes Clostridia Clostridiales Ruminococcaceae Faecalibacterium Firmicutes Clostridia Clostridiales Ruminococcaceae Flavonifractor Firmicutes Clostridia Clostridiales Ruminococcaceae Oscillibacter Firmicutes Clostridia Clostridiales Ruminococcaceae Pseudoflavonifractor Firmicutes Clostridia Clostridiales Ruminococcaceae Ruminococcus Firmicutes Clostridia Clostridiales Ruminococcaceae Subdoligranulum Firmicutes Clostridia Clostridiales Ruminococcaceae ud Ruminococcaceae Firmicutes Clostridia Clostridiales ud Clostridiales ud Clostridiales Firmicutes Clostridia ud Clostridia ud Clostridia ud Clostridia Firmicutes Erysipelotrichia Erysipelotrichales Erysipelotrichaceae Allobaculum Firmicutes Erysipelotrichia Erysipelotrichales Erysipelotrichaceae Catenibacterium Firmicutes Erysipelotrichia Erysipelotrichales Erysipelotrichaceae Clostridium XVIII Firmicutes Erysipelotrichia Erysipelotrichales Erysipelotrichaceae Coprobacillus Firmicutes Erysipelotrichia Erysipelotrichales Erysipelotrichaceae Erysipelotrichaceae_incertae_sedis Firmicutes Erysipelotrichia Erysipelotrichales Erysipelotrichaceae Holdemania Firmicutes Erysipelotrichia Erysipelotrichales Erysipelotrichaceae Solobacterium Firmicutes Erysipelotrichia Erysipelotrichales Erysipelotrichaceae Turicibacter Firmicutes Erysipelotrichia Erysipelotrichales Erysipelotrichaceae ud Erysipelotrichaceae Firmicutes Negativicutes Selenomonadales Acidaminococcaceae Acidaminococcus Firmicutes Negativicutes Selenomonadales Acidaminococcaceae Phascolarctobacterium Firmicutes Negativicutes Selenomonadales Acidaminococcaceae ud Acidaminococcaceae Firmicutes Negativicutes Selenomonadales ud Selenomonadales ud Selenomonadales Firmicutes Negativicutes Selenomonadales Veillonellaceae Allisonella Firmicutes Negativicutes Selenomonadales Veillonellaceae Dialister Firmicutes Negativicutes Selenomonadales Veillonellaceae Megasphaera Firmicutes Negativicutes Selenomonadales Veillonellaceae Mitsuokella Firmicutes Negativicutes Selenomonadales Veillonellaceae Selenomonas Firmicutes Negativicutes Selenomonadales Veillonellaceae ud Veillonellaceae Firmicutes Negativicutes Selenomonadales Veillonellaceae Veillonella Firmicutes ud Firmicutes ud Firmicutes ud Firmicutes ud Firmicutes

7 Bacteroidetes Phylum Class Order Family Genus Frequency (Log scale) Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Barnesiella Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Butyricimonas Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Odoribacter Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Porphyromonas Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae ud Porphyromonadaceae Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae Paraprevotella Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae Prevotella Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae ud Prevotellaceae Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae Xylanibacter Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae Alistipes Bacteroidetes Bacteroidia Bacteroidales Rikenellaceae ud Rikenellaceae Bacteroidetes Bacteroidia Bacteroidales ud Bacteroidales ud Bacteroidales Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae Chryseobacterium Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae Cloacibacterium Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae Flavobacterium Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae ud Flavobacteriaceae Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae Wautersiella Bacteroidetes Sphingobacteria Sphingobacteriales Chitinophagaceae ud Chitinophagaceae Bacteroidetes ud Bacteroidetes ud Bacteroidetes ud Bacteroidetes ud Bacteroidetes V1 V2 V3 V4 V5 V6 Act FS IgA

8 Proteobacteria Phylum Class Order Family Genus Frequency (Log scale) Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae Brevundimonas Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae ud Caulobacteraceae Proteobacteria Alphaproteobacteria Rhizobiales Beijerinckiaceae Beijerinckia Proteobacteria Alphaproteobacteria Rhizobiales Beijerinckiaceae ud Beijerinckiaceae Proteobacteria Alphaproteobacteria Rhizobiales Bradyrhizobiaceae Bradyrhizobium Proteobacteria Alphaproteobacteria Rhizobiales Bradyrhizobiaceae ud Bradyrhizobiaceae Proteobacteria Alphaproteobacteria Rhizobiales Brucellaceae Ochrobactrum Proteobacteria Alphaproteobacteria Rhizobiales Brucellaceae Pseudochrobactrum Proteobacteria Alphaproteobacteria Rhizobiales Brucellaceae ud Brucellaceae Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae Gemmiger Proteobacteria Alphaproteobacteria Rhizobiales Methylobacteriaceae Methylobacterium Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae Mesorhizobium Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae ud Phyllobacteriaceae Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae Rhizobium Proteobacteria Alphaproteobacteria Rhizobiales ud Rhizobiales ud Rhizobiales Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae Paracoccus Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae ud Rhodobacteraceae Proteobacteria Alphaproteobacteria Rhodospirillales Rhodospirillaceae Novispirillum Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae Porphyrobacter Proteobacteria Alphaproteobacteria Sphingomonadales Erythrobacteraceae ud Erythrobacteraceae Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Novosphingobium Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae Sphingomonas Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae ud Sphingomonadaceae Proteobacteria Alphaproteobacteria Sphingomonadales ud Sphingomonadales ud Sphingomonadales Proteobacteria Alphaproteobacteria ud Alphaproteobacteria ud Alphaproteobacteria ud Alphaproteobacteria Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Burkholderia Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Cupriavidus Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Ralstonia Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae ud Burkholderiaceae Proteobacteria Betaproteobacteria Burkholderiales Burkholderiales_incertae_sedis Tepidimonas Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Acidovorax Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Comamonas Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Curvibacter Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Delftia Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Diaphorobacter Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Pelomonas Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Schlegelella Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae ud Comamonadaceae Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae Janthinobacterium Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae Oxalobacter Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae Undibacterium Proteobacteria Betaproteobacteria Burkholderiales Sutterellaceae Parasutterella Proteobacteria Betaproteobacteria Burkholderiales Sutterellaceae Sutterella Proteobacteria Betaproteobacteria Burkholderiales ud Burkholderiales ud Burkholderiales Proteobacteria Betaproteobacteria ud Betaproteobacteria ud Betaproteobacteria ud Betaproteobacteria Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Bilophila Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae Desulfovibrio Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae ud Desulfovibrionaceae Proteobacteria Deltaproteobacteria Desulfovibrionales ud Desulfovibrionales ud Desulfovibrionales Proteobacteria Deltaproteobacteria ud Deltaproteobacteria ud Deltaproteobacteria ud Deltaproteobacteria Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae Aeromonas Proteobacteria Gammaproteobacteria Aeromonadales Succinivibrionaceae Succinivibrio Proteobacteria Gammaproteobacteria Aeromonadales Succinivibrionaceae ud Succinivibrionaceae Proteobacteria Gammaproteobacteria Alteromonadales Shewanellaceae Shewanella Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Buttiauxella Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Cedecea Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Citrobacter Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Escherichia/Shigella Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Hafnia Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Klebsiella Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Morganella Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Obesumbacterium Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Proteus Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Raoultella Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Serratia Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae ud Enterobacteriaceae Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Yersinia Proteobacteria Gammaproteobacteria Legionellales Legionellaceae Legionella Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoracaceae Alcanivorax Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae Halomonas Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae ud Halomonadaceae Proteobacteria Gammaproteobacteria Oceanospirillales ud Oceanospirillales ud Oceanospirillales Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae Actinobacillus Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae Haemophilus Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae ud Pasteurellaceae Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae Acinetobacter Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae Enhydrobacter Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae ud Pseudomonadaceae Proteobacteria Gammaproteobacteria ud Gammaproteobacteria ud Gammaproteobacteria ud Gammaproteobacteria Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae Frateuria Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae Stenotrophomonas Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae ud Xanthomonadaceae Proteobacteria ud Proteobacteria ud Proteobacteria ud Proteobacteria ud Proteobacteria V1 V2 V3 V4 V5 V6 Act FS IgA

9 Actinobacteria Phylum Class Order Family Genus Frequency (Log scale) Actinobacteria Actinobacteria Actinomycetales Actinomycetaceae Actinomyces Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae Corynebacterium Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae ud Corynebacteriaceae Actinobacteria Actinobacteria Actinomycetales Dermabacteraceae Brachybacterium Actinobacteria Actinobacteria Actinomycetales Microbacteriaceae Microbacterium Actinobacteria Actinobacteria Actinomycetales Microbacteriaceae ud Microbacteriaceae Actinobacteria Actinobacteria Actinomycetales Micrococcaceae Micrococcus Actinobacteria Actinobacteria Actinomycetales Micrococcaceae Rothia Actinobacteria Actinobacteria Actinomycetales Nakamurellaceae Nakamurella Actinobacteria Actinobacteria Actinomycetales Nocardiaceae Rhodococcus Actinobacteria Actinobacteria Actinomycetales Nocardiaceae ud Nocardiaceae Actinobacteria Actinobacteria Actinomycetales Nocardioidaceae Nocardioides Actinobacteria Actinobacteria Actinomycetales Nocardioidaceae ud Nocardioidaceae Actinobacteria Actinobacteria Actinomycetales Propionibacteriaceae Propionibacterium Actinobacteria Actinobacteria Actinomycetales Sanguibacteraceae Sanguibacter Actinobacteria Actinobacteria Actinomycetales ud Actinomycetales ud Actinomycetales Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae ud Bifidobacteriaceae Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Collinsella Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Eggerthella Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Gordonibacter Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae Olsenella Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae ud Coriobacteriaceae Actinobacteria Actinobacteria Rubrobacterales Rubrobacteraceae Rubrobacter Actinobacteria Actinobacteria ud Actinobacteria ud Actinobacteria ud Actinobacteria V1 V2 V3 V4 V5 V6 Act FS IgA

10 Figure 3: Control flow cytometry bi-plots. Top panel shows size (X axis) versus complexity (Y axis) of unstained cells compared with 3, 6, and 10 micrometer beads. In bottom panel X axis shows fluorescence emitted by DNA labelled with SYTO62 dye. Y axis represents the fluorescence emitted by anti-s-iga immunoglobulin labelled with FITC. Negative control panel reports unstained cells. Isotype control reports cell stained with SYTO62 (DNA) and mouse-anti-iga as isotype control in order to identify unspecific hybridisation region. Experiment plot shows the human-anti-s-iga hybridised cells. Green area indicates the unspecific region obtained by isotype control.

11 Figure 4: Flow Cytometry bi-plot. X axis show fluorescence emitted by DNA labelled with Syto62 dye reported in arbitrary logarithmic units. First two columns show respectively control experiments without staining (Negative), and only DNA staining (Syto62) visible as an increment on the X axis. Third and fourth columns show the fluorescence emitted by anti-s-iga staining against: mouse (isotype control) and human (experiment for sorting) respectively. S-IgA is shown on the Y axis while DNA staining is represented on the X axis. The fifth column reports pyronin-y staining (Y axis) as a measure of cell activity. Every line corresponds to a volunteer sample. Three of the experimental files have been lost due to technical IT problems (NAs).